Prostaglandin D synthase-specific monoclonal antibody

ABSTRACT

The invention relates to a monoclonal antibody specifically recognizing lipocalin-type prostaglandin D synthase (L-PGDS), a hybridoma producing said monoclonal antibody, methods for detection of L-PGDS or diseases by said monoclonal antibody, and a kit for detection of L-PGDS by said monoclonal antibody. According to the invention, there is provided a monoclonal antibody specific to L-PGDS.

FIELD OF THE INVENTION

[0001] The present invention relates to a monoclonal antibody specificto human L-PGDS present predominantly in cerebrospinal fluid (CSF), ahybridoma producing said monoclonal antibody, methods for detection ofL-PGDS or diseases by said monoclonal antibody, and a kit for detectionof L-PGDS by said monoclonal antibody.

BACKGROUND OF THE INVENTION

[0002] Prostaglandin D is a biologically active substance synthesized inanimal tissues from arachidonic acid released from a biomembrane uponstimulation, and it is produced from prostaglandin H (a common precursorof prostaglandin families produced by cyclo-oxygenase) by prostaglandinD synthase (PGDS).

[0003] The presence of two types of PGDS, glutathione-independent L-PGDSand glutathione-dependent spleen type PGDS, is known (Shimizu et, al.,J. Biol. Chem., 25A, 5222-5228 (1979); Urade et al., J. Biol. Chem.,260, 12410-12415 (1985); Christ-Hazelhof and Nugteren, Biochim. Biophys.Acta, 572, 43-51 (1979); Urade et al., J. Biol. Chem., 262, 3820-3825(1987). The former is known to be predominantly located in the centralnervous system such as brain, epididymis, spinal cord, retina, and innerear (Urade et al., J. Biol. Chem., 26, 12410-12415 (1985); Ueno et al.,J. Neurochem., 45, 483-489 (1985); Urade et al., J. Biol. Chem., 2,3820-3825 (1987); Goh et al., Biochim. Biophys. Acta, 921, 302-311(1987); and Tachibana et al., Proc. Natl. Acad. Sci. USA, 84, 7677-7680(1987)), and the latter is known to be distributed broadly in almost allperipheral organs including spleen, bone marrow, digestive organs,thymus, and skin (Ujihara et al., Arch. Biochem. Biophys., 260, 521-531(1988); and Ujihara et al., J. Invest., Dermatol., 90, 448-451 (1988)).

[0004] On the other hand, a protein called β-trace was observed to bepresent specifically in human CSF, but its physiological functionremained unrevealed (Causen, J. Proc. Soc. Exp. Biol. Med., 107, 170-172(1961)).

[0005] A certain correlation between β-trace known as a protein specificto CSF and severe brain disorders or certain diseases (multiplesclerosis, brain tumors, Meckel's syndrome and paraproteinemia) wasnoted from the observation that β-trace levels depend on such disorders(Ericsson et al., Neurology, 19, 606-610 (1969); Olsson et al., J.Neurol. Neurosurg. Psychiat. 37, 302-311 (1974); Link, J. Neurol. Sci.,16, 103-114 (1972); Whistsed and Penny, Clinica Chimica Acta, 50,111-118 (1974); and Chemke et al., Clinical Genetics, 11, 285-289(1977)). However, the exact correlation between β-trace and suchdisorders could not be determined because the physiological function ofβ-trace still remained unrevealed and because there was no toolavailable for determining the exact amount (concentration) of β-trace.

[0006] Recently, the nucleotide sequence of cDNA coding for L-PGDS wasreported (Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024(1991)), and production of L-PGDS by genetic recombination becamefeasible. The L-PGDS thus produced was examined and its amino acidsequence was estimated and compared with an N-terminal partial aminoacid sequence of human β-trace in searching for their homology(Kuruvilla et al., Brain Research, 56, 337-340 (1991), Zahn et al.,Neuroscience Letters, 154, 93-95 (1993)) or with the amino acid sequenceof purified human β-trace (Hoffmann et al., J. Neurochem., 61(2),451-456 (1993)), and further immunological examination was made usingpolyclonal antibodies (Watanabe et al., Biochem. Biophys. Res.Communication, 203, 1110-1116 (1994)). These studies revealed thatβ-trace was identical with L-PGDS.

[0007] Prostaglandin D occurring abundantly in the central nervoussystem functions, in one physiological action, as a neuromodulator ofseveral central actions including sleep promotion. Prostaglandin Dsynthase is considered as a key enzyme for sleep-wake activities(Hayashi, FASEB J., 5, 2575-2581 (1991)), and it is believed that atleast a part of the L-PGDS secreted from competent cells is accumulatedin CSF (Watanabe et al., Biochem. Biophys. Res. Communication, 203,1110-1116 (1994)).

[0008] Accordingly, the analysis of L-PGDS distribution etc. in thecentral nervous system is useful for detection of diseases in thecentral nervous system, and it is expected that L-PGDS levels in CSF orhumor can also be used as an indicator in early diagnosis and prognosticobservations for other diseases caused by abnormalities in the centralnervous system. It is further expected that L-PGDS (or β-trace) can beused for examination of a reproduction ability, diagnosis of fetalgrowth, etc. because this enzyme is distributed in such humors derivedfrom genital organs, as semen, oviduct fluid and amniotic fluid, aswell. For such applications, there is demand for antibodies specificallyrecognizing L-PGDS.

[0009] Nevertheless, such antibodies have still not been establishedwith high specificity to meet such demand.

DISCLOSURE OF THE INVENTION

[0010] The object of the present invention is to provide a monoclonalantibody specifically recognizing L-PGDS, a hybridoma producing saidantibody, methods for detection of L-PGDS or diseases by said monoclonalantibody, and a kit for detection of L-PGDS by said monoclonal antibody.

[0011] As a result of their eager researches, the present inventors havesuccessfully obtained a monoclonal antibody specifically recognizingL-PGDS from a hybridoma prepared by cell fusion between myeloma cellsand antibody-producing cells from an animal immunized with L-PGDS whichis a major protein in human CSF, and they thereby arrived at the presentinvention.

[0012] That is, the present invention relates to a monoclonal antibodyspecifically recognizing L-PGDS. The subclass of such monoclonalantibody includes immunoglobulin G1 or G2.

[0013] Further, the present invention relates to a hybridoma producingsaid monoclonal antibody by cell fusion between myeloma cells andantibody-producing cells from an animal immunized with L-PGDS

[0014] Further, the present invention relates to methods for detectionof L-PGDS or diseases by said monoclonal antibody. An example of suchdiseases is oligospermia.

[0015] Further, the present invention relates to a kit for detection ofL-PGDS, which is selected from a reagent containing said monoclonalantibody labeled with an enzyme and a substrate solution, and a reagentcontaining said monoclonal antibody obtained by biotination,enzyme-labeled avidin, and a substrate.

[0016] Further, the present invention relates to a method for detectionof diseases by said kit for detection of L-PGDS. An example of suchdiseases is oligospermia.

[0017] Hereinafter, the present invention is described in detail.

[0018] 1. Production of the Monoclonal Antibody

[0019] Production of the present monoclonal antibody against L-PGDSconsists of the steps of:

[0020] (1) Preparation of an antigen;

[0021] (2) Immunization and preparation of antibody-producing cells;

[0022] (3) Establishment of an antibody-titration system;

[0023] (4) Cell fusion;

[0024] (5) Selecting and cloning hybridomas; and

[0025] (6) Isolation of the monoclonal antibody.

[0026] Hereinafter, each step is described.

[0027] (1) Preparation of an Antigen

[0028] L-PGDS can be produced in large amounts in a usual manner by E.coli and CHO cells etc. with its known cDNA (Nagata et al., Proc. Natl.Acad. Sci. USA, 88, 4020-4024 (1991)). In this production of L-PGDS, arecombinant DNA containing the cDNA for L-PGDS is constructed andtransformed into a microorganism, which is then cultured to produce theenzyme. The L-PGDS thus produced can be purified from the culture byconventional means.

[0029] Then, an immunogen is prepared by dissolving the resulting L-PGDSin a buffer and then adding an adjuvant to it. Examples of suchadjuvants are Freund complete adjuvant, Freund incomplete adjuvant, BCG,Hunter's Titermax (CytRx Corporation), key hole limpethemocyanin-containing oil, etc., and any of them can be mixed.

[0030] (2) Immunization and Preparation of Antibody-producing Cells

[0031] The immunogen thus obtained is administrated as antigen intomammals such as horse, monkey, dog, pig, cow, goat, sheep, rabbit,guinea pig, hamster and mouse, or birds such as pigeon and chicken. Inparticular, mouse, rat, guinea pig, rabbit and goat are preferably used.Any of the known immunization methods may be employed preferably usingi.v., s.c., or i.p. administration. Immunization intervals are notparticularly limited, and the immunogen is given 2 to 10 times,preferably 2 to 5 times, preferably at intervals of several days toseveral weeks, more preferably 1 to 3 weeks.

[0032] 1 to 10 days preferably 2 to 5 days after the final immunization,antibody-producing cells are prepared from the animal. Examples of suchantibody-producing cells are spleen cells, lymph node cells, thymocytesand peripheral blood cells, and generally spleen cells are usedconventionally. In the case of mouse, 0.01 μg to 1,000 μg, preferably 1to 300 μg antigen is given per animal in one administration.

[0033] (3) Establishment of an Antibody-titration System

[0034] It is necessary to establish a system of measuring the antibodytiter in serum from the immunized animal or in a culture supernatantfrom the antibody-producing cells, so that the immune response level ofthe immunized animal can be confirmed and the desired hybridoma can beselected from the fusion cells. For example, the antibody can bedetected conventionally using known methods such as enzyme immunoassay(EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA)and fluorescence immunoassay (FIA). Although the present method fordetecting the antibody is not limited to the above methods, ELISA isconveniently used for easy operations. Usually, L-PGDS is put to eachwell of a 96-well plastic microtiter plate and left at room temperatureto immobilize the enzyme onto the well. Then, the unbound sites of theL-PGDS as antigen are blocked with calf serum albumin, fetal bovineserum, skim milk, or gelatin. Then, an antiserum diluted with phosphatebuffered saline (referred to hereinafter as PBS) or a culturesupernatant of the hybridoma is added to each well. Subsequently,commercial secondary antibody labeled with an enzyme or fluorescentcompound or with biotin is added to each well, followed by adding acoloration substrate. The coloration occurring can be determined in aphotometer or fluorometer to quantify the antibody against L-PGDS.

[0035] (4) Cell Fusion

[0036] The myeloma cells to be subjected to cell fusion with theantibody-producing cells are those derived from animal species such asmouse, rat, and human, easily available to those skilled in the art. Thecell line used is preferably drug-resistant, which upon fusion with theantibody-producing cells, is rendered survivable in a selection mediumsuch as HAT medium. A cell line resistant to 8-azaguanine is generallyused. Because such cell line lacks hypoxanthine-guaninephosphoribosyltransferase (HGPRT-), it can not grow inhypoxanthine-aminopterine-thymidine (HAT) medium. The cell line used ispreferably cells not secreting immunoglobulins.

[0037] Examples of such myeloma cells are mouse myeloma cell lines suchas P3X63Ag8 (ATCC TIB-9; Nature, 256, 495-497 (1978)), P3X63Ag8U.1(P3U1) (ATCC CRL-1580; Current Topics in Microbiology and Immunology,al, 1-7 (1978), P3X63Ag8.653 (ATCC TIB-18; European J. Immunology, 6,511-519 (1976)), and P2/NSI/1-Ag4-1 (ATCC CRL-1581; Nature, 276, 269-270(1978)); rat myeloma cell lines such as for example 210. RCY.Agl.2.3(Y3-Agl.2.3) (ATCC CRL-1631; Nature, 277, 131-133 (1979)); human myelomacell lines such as U-266-AR1 (Proc. Natl. Acad. Sci. USA, 77, 5429(1980)), GM 1500 (Nature, 288, 488 (1980)), and KR-4 (Proc. Natl. Acad.Sci. USA, 79, 6651 (1982)).

[0038] The antibody-producing cells are obtained from spleen cells,lymph node cells, thymocytes, and peripheral blood cells. Briefly, theantibody-producing cells are prepared as follows: Tissues such asspleen, lymph node or thymus are excised or blood are collected from theimmunized animals. These tissues are disrupted and then suspended in abuffer such as PBS or in a medium such as DMEM, RPMI 1640 and E-RDF.This cell suspension is filtered through e.g. a #200-250 stainless meshand then centrifuged to give the desired antibody-producing cells.

[0039] Then, the antibody-producing cells are subjected to cell fusionwith myeloma cells.

[0040] Before cell fusion, myeloma cells suitable for antibodyproduction are selected. 10⁶ to 10⁸ cells/ml antibody-producing cellsare mixed with 10⁶ to 10⁸ cells/ml myeloma cells at a ratio of from 1:1to 1:10 in an animal cell growth medium, e.g. Eagle's minimal essentialmedium (MEM), Dulbecco's modified Eagle's medium (DMEM), RPMI-1640medium, or E-RDF medium. To enhance cell fusion, these cells are mixedat a ratio of e.g. 1:6 and then incubated for 1 to 15 minutes in thepresence of a fusion enhancer in e.g. RPMI-1640 medium containingdimethylsulfoxide at a temperature of 30 to 37° C. This fusion enhancermay be polyethylene glycol with an average molecular weight of 1,000 to6,000, polyvinyl alcohol or Sendai virus. Alternatively, theantibody-producing cells can be subjected to cell fusion with myelomacells by electric stimulation (e.g. electroporation) in a commercialcell fusion apparatus.

[0041] (5) Selection and Cloning of Hybridomas

[0042] After cell fusion, the cells are screened for desired hybridomas.The selective growth of the fusion cells in a selection medium may beused for screening as follows: The cell suspension is diluted 5- to10-fold with e.g. E-RDF medium containing 15% fetal bovine serum andthen put to each well of a microtiter plate at about 10² to 10⁶cells/well, followed by addition of a selection medium (e.g. HAT medium)to each well. Thereafter, the cells are incubated while the selectionmedium is exchanged with fresh one at suitable intervals.

[0043] Where myeloma cells are from an 8-azaguanine-resistant strain andHAT medium is used as selection medium, the myeloma cells andantibody-producing cells, if they fail to fuse, will die during in vitroculture in about 7 days and antibody-producing cells in 10 days. Hence,hybridomas can be obtained from those cells beginning to grow after the10th day of culture.

[0044] The hybridomas are screened for the desired ones by examiningtheir supernatants on the presence of the antibodies against L-PGDS.This screening step can be carried out by any of the conventionalmethods. For example, a supernatant (first antibody) from hybridomasgrown in each well is put to a well with L-PGDS immobilized on it, thena labeled secondary antibody is added to the well and incubated, and thebinding ability of the secondary antibody is then examined in enzymeimmunoassays (EIA, ELISA), RIA, etc.

[0045] In more detail, the screening of hybridomas is carried out asfollows: Natural or recombinant L-PGDS, which was used as immunogen, isimmobilized as antigen onto a 96-well microtiter plate. A culturesupernatant expected to contain the monoclonal antibody is added to eachwell and reacted with the immobilized antigen. Then, the antigen-boundmonoclonal antibody, if any, is reacted with another antibody(enzyme-labeled anti-immunoglobulin antibody). Alternatively, saidimmobilized monoclonal antibody is reacted with a biotinylatedanti-immunoglobulin antibody and then with enzyme-labeled avidin.Finally, each well is colored by adding an enzyme substrate solution.The hybridomas whose culture supernatants are colored in the wellshaving the immobilized natural or recombinant L-PGDS are those producingantibodies having the ability to bind to the L-PGDS.

[0046] These hybridomas can be cloned in conventional methods includinglimiting dilution, soft agar cloning, fibrin gel cloning andfluorescence excitation cell sorting to give the desired monoclonalantibody-producing hybridoma.

[0047] (6) Isolation of the Monoclonal Antibody

[0048] From the resulting hybridoma, the monoclonal antibody can beisolated using conventional methods such as cell culture method, ascitestransudate method, etc.

[0049] In the cell culture method, the hybridoma is cultured for 2 to 14days in a medium such as RPMI-1640, MEM, or E-RDF containing 10 to 20%calf serum or in a serum-free medium under conventional cultureconditions, e.g. 37° C., 5% CO₂. The antibody can be obtained from theculture.

[0050] In the ascites transudate method, a mineral oil such as pristane(2,6,10,14-tetramethylpentadecane) is administrated by i.p. to the samemammal species as the mammal from which the myeloma cells were derived.Then, the hybridoma, 1×10⁷ to 1×10⁹ cells, preferably 5×10⁷ to 1×10⁸cells, are administrated by i.p. to the animal, and a large amount ofhybridoma cells are grown in the animal. After 1 to 4 weeks, preferably2 to 3 weeks, ascites fluid or serum is collected from the animal.

[0051] If it is necessary to purify the antibody from the ascites fluidor serum, it can be purified by conventional methods such as salting-outwith ammonium sulfate, ion-exchange chromatography on anion exchangere.g. DEAE cellulose, affinity chromatography on Protein A Sepharose, andgel filtration, and these may be used singly or in combination.

[0052] 2. Method of Detecting L-PGDS by the Monoclonal Antibody of thePresent Invention

[0053] The method of detecting L-PGDS according to the present inventioncan be carried out using said monoclonal antibody, as follows:

[0054] A 96-well microtiter plate is coated with a diluted sample suchas CSF, serum etc. and then blocked with e.g. 0.2% gelatin in PBS. Then,the monoclonal antibody of the present invention, labeled with anenzyme, is added to each well and then incubated; alternatively, themonoclonal antibody labeled with biotin is added to each well, then theplate is washed, enzyme-labeled avidin or streptoavidin is added to eachwell, and the plate is further incubated. Then, the plate is washed anda coloration substrate such as ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)) is added toeach well. L-PGDS can be determined by examining this coloration in thecolorimetric method.

[0055] In another embodiment of the present invention, a 96-wellmicrotiter plate is coated with the diluted monoclonal antibody of thepresent invention and then blocked with e.g. 0.2% gelatin in PBS. Then,a diluted sample such as CSF, serum etc. is added to each well and theplate is incubated. After washing the plate, another enzyme-labeledmonoclonal or polyclonal antibody solution is added to each well and theplate is incubated; alternatively, the monoclonal antibody or polyclonalantibody labeled with biotin is added to each well, then the plate iswashed, enzyme-labeled avidin or streptoavidin is added to each well,and the plate is further incubated. Then, the plate is washed and acoloration substrate such as ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)) is added toeach well. The L-PGDS can be determined by examining this coloration inthe calorimetric method. In this manner, it is possible to detect andquantify L-PGDS.

[0056] 3. A Reagent for Measurement of L-PGDS by the Monoclonal Antibodyof the Present Invention

[0057] The monoclonal antibody of the present invention is useful as areagent for measurement of L-PGDS because it specifically binds toL-PGDS. Furthermore, the present monoclonal antibody is useful as areagent in determining the presence and distribution of not only L-PGDSas antigen but also other similar antigens having the same epitope asthat of L-PGDS, as well as fragments of L-PGDS in biological samplessuch as organs, tissues, cells and humor. Hence, the antibody of thepresent invention is useful as a reagent for such measurement anddiagnosis. The detection or measurement of L-PGDS in organs, tissues,cells and humor can be effected using quantitative or qualitative meanssuch as EIA, ELISA, RIA, FIA, Western blot technique andimmunohistochemistry, etc.

[0058] 4. A Kit for Detection of L-PGDS

[0059] The kit of the present invention, if an enzyme is used as a labelfor detection, contains the following ingredients:

[0060] (1) monoclonal antibody labeled with an enzyme; and

[0061] (2) substrate.

[0062] The kit of the present invention, if modified with the sandwichELISA method, contains the following ingredients:

[0063] (1) monoclonal antibody;

[0064] (2) monoclonal or polyclonal antibody labeled with an enzyme; and

[0065] (3) substrate.

[0066] The kit of the present invention, if modified with thebiotin-avidin method, contains the following ingredients:

[0067] (1) biotinated monoclonal antibody;

[0068] (2) enzyme-labeled avidin or streptoavidin; and

[0069] (3) substrate.

[0070] The kit of the present invention, if modified with the sandwichELISA and biotin-avidin methods, contains the following ingredients:

[0071] (1) monoclonal antibody;

[0072] (2) biotinated monoclonal or polyclonal antibody;

[0073] (3) enzyme-labeled avidin or streptoavidin; and

[0074] (4) substrate.

[0075] In the ingredients, the “monoclonal antibody” means themonoclonal antibody of the present invention. The “polyclonal antibody”means an antibody contained in serum from an animal immunized withL-PGDS, and it can be prepared in the following manner.

[0076] (1) Preparation of the Antigen

[0077] L-PGDS can be produced in large amounts in a usual manner by E.coli and CHO cells etc. with its known cDNA (Nagata et al., Proc. Natl.Acad. Sci. USA, 88, 4020-4024 (1991)). For production of L-PGDS, arecombinant DNA containing the cDNA for L-PGDS is constructed andtransformed into a microorganism, and the transformant is cultured toproduce the enzyme. The resulting L-PGDS can be purified from theculture by conventional means.

[0078] L-PGDS thus obtained is dissolved in a buffer, and an immunogenis prepared by adding an adjuvant to it. Examples of adjuvants areFreund complete adjuvant, Freund incomplete adjuvant, BCG, Hunter'sTitermax (CytRx Corporation), key hole limpet hemocyanin-containing oil,etc., and any of them can be mixed.

[0079] (2) Immunization and Preparation of Blood

[0080] The immunogen thus obtained is administrated into mammals such ashorse, monkey, dog, pig, cow, goat, sheep, rabbit, guinea pig, hamsterand mouse, or birds such as pigeon and chicken, among which mouse, rat,guinea pig, rabbit and goat are preferably used. Any of the knownimmunization methods can be employed preferably via i.v., s.c., or i.p.administration. Immunization intervals are not particularly limited, andthis administration is carried out 2 to 10 times, preferably 2 to 5times, at intervals of preferably several days to several weeks, morepreferably 1 to 3 weeks.

[0081] Antibody titer in blood from the immunized animal is determinedaccording to the above method in (3) “1. Production of the monoclonalantibody”. Blood samples found to have high antibody titer are left atroom temperature or 4° C. and centrifuged to give serum containing thepolyclonal antibody.

[0082] If it is necessary to purify the polyclonal antibody from theserum, it can be purified by conventional methods such as salting-outwith ammonium sulfate, ion-exchange chromatography on anion exchangere.g. DEAE cellulose, affinity chromatography on Protein A Sepharose, andgel filtration separating molecules depending on molecular weight andstructure, and these may be used in singly or in combination.

[0083] According to the present invention, various diseases can bedetected by use of the present kit for detection of L-PGDS. For example,oligospermia can be diagnosed readily and rapidly by the present kitusing the monoclonal antibody.

[0084] The monoclonal antibody of the present invention can also be usedto purify L-PGDS. That is, the monoclonal antibody of the presentinvention is coupled in a usual manner to carriers such as agarose,cellulose, acrylamide gel, commercially available self-made affinitycarriers and then washed. L-PGDS can be purified easily with high yieldby elution from the column with a suitable solvent or buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0085]FIG. 1 is a photograph showing a profile in SDS-PAGE.

[0086]FIG. 2 is a photograph showing a profile in SDS-PAGE and Westernblotting of CSF and purified L-PGDS.

[0087]FIG. 3 is a photograph showing a profile in Western blotting ofpurified L-PGDS treated with N-glycanase.

[0088]FIG. 4 shows the result of an epitope mapping of each monoclonalantibody.

[0089]FIG. 5 is a calibration curve for L-PGDS (by monoclonal antibody1B7).

[0090]FIG. 6 is a calibration curve for L-PGDS (by monoclonal antibody6F5).

[0091]FIG. 7 is a calibration curve for L-PGDS (by sandwich ELISA methodusing monoclonal antibody 1B7 as primary antibody and biotinylatedpolyclonal antibody as secondary antibody).

[0092]FIG. 8 is a calibration curve for L-PGDS (by sandwich ELISA methodusing monoclonal antibody 6F5 as primary antibody and biotinylatedpolyclonal antibody as secondary antibody).

[0093]FIG. 9 is a calibration curve for L-PGDS (by sandwich ELISA methodusing monoclonal antibody 1B7 as primary antibody and biotinylatedpolyclonal antibody (A), biotinylated 7B5 (B) and biotinylated 10A3 (C)as secondary antibodies).

[0094]FIG. 10 is a calibration curve for L-PGDS (by sandwich ELISAmethod using monoclonal antibody 10A3 as primary antibody andbiotinylated polyclonal antibody (A) and biotinylated 1B7 (B) assecondary antibodies).

[0095]FIG. 11 is a calibration curve for L-PGDS (by sandwich ELISAmethod using monoclonal antibody 7F5 as primary antibody andbiotinylated polyclonal antibody (A) and biotinylated 1B7 (B) assecondary antibodies).

[0096]FIG. 12 is a drawing showing a calibration curve prepared usingL-PGDS and calibration curves for L-PGDS which were prepared usingseveral samples.

[0097]FIG. 13 is a photograph showing a profile in Western blotting.

[0098]FIG. 14 shows the result of measurement of L-PGDS.

[0099]FIG. 15 is a photograph showing a profile in SDS-PAGE of L-PGDSpurified by monoclonal antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0100] Hereinafter, the present invention is described in more detail byreference to Examples. However, the present invention is not limited tothe Examples.

Example 1 Production of the Monoclonal Antibody

[0101] (1) Preparation of the Antigen

[0102] L-PGDS was prepared as antigen by genetic engineering.

[0103] A GST gene fusion system (Pharmacia) was used for expression ofthe antigen in E. coli and purification. The following procedure wascarried out for fusion of L-PGDS with GST protein.

[0104] A 185 bp product was obtained by amplifying a region of cDNAcoding for N-terminal region of L-PGDS by polymerase chain reaction(PCR) in the following manner.

[0105] The primer nucleotide sequences used are:

[0106] Ec23ALA: Sequence ID NO:1.

[0107] 78NMUTA: Sequence ID NO:2.

[0108] PCR was carried out using Taq DNA polymerase (Takara Shuzo Co.,Ltd.), restriction enzymes EcoRI and XhoI, and T4 DNA ligase (TakaraShuzo) where 1 cycle reaction (94° C. for 5 seconds, 45° C. for 3seconds, and at 72° C. for 5 seconds) was repeated 28 times.

[0109] With these primers, the partial nucleotide sequence between the152 to 327 positions (from guanine (G) to cytosine (C), corresponding tothe partial amino acid sequence between the N-terminal (alanine) and the81 position (serine) on the amino acid sequence of the mature proteinexcluding its signal sequence) was amplified and an EcoRI site wasintroduced into the 5′-terminal. Because this PCR product had an XhoIsite at the 238 position, the product was subcloned by digestion withrestriction enzymes EcoRI and XhoI. A recombinant DNA was obtained byreplacement, by the subcloned product, of the corresponding N-terminalregion of the cDNA for native L-PGDS (Nagata et al., Proc. Natl. Acad.Sci. USA, 88, 4020-4024 (1991)). This recombinant DNA was inserted intoan EcoRI site of vector pGEX-2T (Pharmacia) which was selected for GSTfusion protein.

[0110] Alternatively, a 521 bp product was obtained by amplifying thatregion of cDNA coding for the whole mature protein of L-PGDS by PCR asdescribed below.

[0111] The primer nucleotide sequences used are:

[0112] forward primer: Sequence ID NO:3.

[0113] reverse primer: Sequence ID NO:4.

[0114] PCR was carried out using Taq DNA polymerase (Takara Shuzo) where1 cycle reaction (94° C. for 5 seconds, 45° C. for 3 seconds, and at 72°C. for 5 seconds) was repeated 28 times.

[0115] With these primers, the region.between the 152 to 656 positions(from guanine (G) to adenine (A), corresponding to the region betweenthe N-terminal (alanine) to the C-terminal (glutamine) of the amino acidsequence of the mature protein excluding its signal sequence) wasamplified, and a BamHI site was introduced at the 5′-terminal and anEcoRI site at the 3′-terminal. The amplified DNA was inserted intoEcoRI/BamHI sites of vector pGEX-2T (Pharmacia) for GST fusion protein.

[0116] The resulting expression vector for GST-L-PGDS fusion protein wastransformed in a usual manner into E. coli DH5α or JM109. The fusionprotein produced by the transformant was recovered by selectiveabsorption onto affinity chromatography beads (Pharmacia) and subsequentelution with thrombin according to manufacture's instructions. In thismanner, about 2 mg L-PGDS was obtained from 100 ml culture of thetransformant.

[0117] Proteins produced by 2 independent clones (E. coli DH5α/pGDS2 andE. coli DH5α/pGDS7) were analyzed by SDS-PAGE on 10-20% gradient gel.The results are shown in FIG. 1.

[0118] In FIG. 1, lane 1 shows bands of molecular-weight markers; lanes2 to 5, from one of the above clones; lanes 6 to 9, from the otherclone; lanes 2 and 6, homogenates of the respective clones; lanes 3 and7, fractions not absorbed onto the affinity column; lanes 4 and 8,fractions eluted with thrombin; and lanes 5 and 9, fractions eluted witha buffer containing reduced glutathione. In lanes 2 and 6, a bandcorresponding to a molecular weight of about 45 kDa is the fusionprotein, and this band is scarcely observed in the GST-unbound fractions(lanes 3 and 7). The fractions eluted with glutathione (lanes 5 and 9)showed the fusion protein band and GST band (molecular weight of about25 kDa), which were not eluted with thrombin.

[0119] According to these results in SDS-PAGE, L-PGDS was produced in E.coli in the form of an about 45 kDa fusion protein with GST, and theprotein with a molecular weight of about 20 kDa, eluted with thrombin(lanes 4 and 8), is L-PGDS itself because this molecular weightcorresponds to the molecular weight of 20 kDa deduced from thenucleotide sequence for L-PGDS. (2) Preparation of theantibody-producing cells A 0.5 ml solution containing 500, μg L-PGDSobtained in (1) was mixed with 0.5 ml Freund complete adjuvant andemulsified for 3 to 5 minutes. As antigen, 100 μl of the emulsion wasadministrated by s.c. into the tail rump of a BALB/c mouse. 3 weeksafter the first immunization, the same volume of another antigenemulsion in Freund incomplete adjuvant was administrated by i.p. to themouse for boosting. 3 weeks after the second immunization, 100 μgantigen (100 μg antigen/200 μl PBS) was administrated to each mouse viaits tail vein. 3 days after the final immunization, the spleen wasexcised from the immunized mouse and disrupted in E-RDF medium to give acell suspension.

[0120] (3) Cell Fusion

[0121] The suspended 1×10⁸ spleen cells were subject to cell fusion with1×10⁷ mouse myeloma cells P3-X63-Ag8-U1 (P3-U1) or P3-X-63-Ag8.653 in50% (W/V) PEG (molecular weight 1,500, Boehringer Mannheim) according tothe method of Oi and Herzenberg (Selected Methods in CellularImmunology, 351-371, W. H. Freeman & Co., USA press, 1980).

[0122] (4) Selection of the Hybridoma

[0123] According to the above-mentioned method of Oi et al., the desiredhybridoma was selected in HAT medium, i.e. E-RDF medium containing 1.36mg/dl hypoxanthine, 19.1 μg/dl aminopterin, 387 μg/dl thymidine, 10%fetal bovine serum and 5% Origen HCF (IGEN).

[0124] (5) Selection of the Monoclonal Antibodies

[0125] The antibody-positive cells in 15 wells were cloned by repeatinglimiting dilution at least twice. The resulting 6 clones were culturedto give strains producing a significant amount of the monoclonalantibody specific to L-PGDS, as follows:

[0126] 1×10⁷ hybridoma cells were cultured at 37° C. for 4 days in 5%Co₂ in the 225 cm² flask containing 50 ml E-RDF medium with 10% fetalcalf serum. Among the resulting 6 cell lines, 5 lines were selected. Theresults are shown in Table 1. TABLE 1 number of number of cell number ofantibody- number of antibody Antigen total wells growth wells positivewells established wells hPGDS 960 778 15 6

[0127] In Table 1, “number of cell growth wells” means the number ofwells where hybridomas could grow in selective culture in HAT medium;“number of antibody-positive wells”, the number of wells where antibodyproduction was detected by ELISA using the antigen prepared in Example 1(1); and “number of antibody established wells”, the number of wellswhere hybridomas producing the specific antibody were established bycloning. The 5 selected cell lines were designated 1B7, 6F5, 7F5, 9A6and 10A3, respectively. The antibodies produced by these cell lines weregiven the same designations as above. The cell lines 1B7, 6F5, 7F5, 9A6and 10A3 have been deposited as FERM BP-5709 (original deposit date:September 21, 1995), FERM BP-5710 (original deposit date: September 21,1995), FERM BP-5711 (original deposit date: June 6, 1996), FERM BP-5712(original deposit date: June 6, 1996) and FERM BP-5713 (original depositdate: Jun. 6, 1996), respectively, with National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology(Higashi 1-1-3, Tsukuba City, Ibaragi Pref., Japan).

[0128] (6) Production of the Monoclonal Antibodies

[0129] Each of the cell lines 1B7, 6F5, 7F5, 9A6 and 10A3, selectedabove in (5), was administrated by i.p. to mice. Remaining resultingcell strain, 6B9 was also administrated by i.p. to mice. Briefly, 1 mlpristane was administrated by i.p. to each mouse. 2 weeks thereafter, 1X10⁸ hybridoma cells were inoculated intraperitoneally to the mouse, and2 weeks thereafter, the transuded ascites was collected from the mouse.

[0130] The collected ascites was applied to Protein A affinity columnchromatography according to a conventional method.

[0131] As a result, 3 to 10 mg/ml antibodies against L-PGDS wereobtained.

[0132] Typing of the antibodies of 1B7, 6F5 with Isotyping Kit (RPN29,AMERSHAM) indicated that all of them belong to IgGl subclass and possessλ light chain.

EXAMPLE 2 Properties of the Monoclonal Antibodies

[0133] (1) Specificity Examination by ELISA

[0134] A 96-well microtiter plate was coated with various types ofL-PGDS and blocked with 0.2% gelatin in PBS. After blocking, each of the6 antibodies obtained in Example 1 (5) was examined for specificity inELISA after adding 50 μl antibody solution to each well. The results areshown in Table 2. TABLE 2 antigen 1B7 6B9 6F5 7F5 9A6 10A3 E. coliantigen ∘ ∘ ∘ ∘ ∘ ∘ E. coli lysate x x x x x x CHO antigen ∘ ∘ ∘ ∘ x ∘CSF antigen ∘ ∘ ∘ x x x

[0135] The results in Table 2 suggested that at least 3 antibodies 1B7,6B9 and 6F5 specifically recognize L-PGDS itself.

[0136] (2) Specificity Examination by Western Blotting

[0137] The above antigens were electrophoresed by SDS-PAGE on 16%isocratic gel and then subjected to Western blot analysis using 5 kindsof antibody. The results are shown in Table 3. TABLE 3 antigen 1B7 6B96F5 9A6 10A3 E. coli antigen ∘ ∘ ∘ ∘ ∘ ∘ E. coli lysate x x x x x CHOantigen ∘ ∘ ∘ x O

[0138] A signal, appearing in an immunoblot profile obtained incoloration after Western blotting, was detected at a positioncorresponding to the molecular weight (about 20 to 30 kDa) ofprostaglandin D synthase. Hence, the antigens showing such signals areconsidered specific to prostaglandin D synthase.

[0139]FIG. 2 shows the results of Western blot analysis using CSFantigen and CSF itself.

[0140] Each lane A shows CSF where 2 μl was applied. Each lane B showsL-PGDS (CSF antigen) purified from CSF where 50 ng was applied. Silverstaining indicated a large number of proteins in CSF, and the purifiedL-PGDS appears as a broad band with a molecular weight of about 27 kD.

[0141] Each monoclonal antibody was used in Western blotting. Theresults revealed that all monoclonal antibodies (1B7, 6B9, 6F5, 7F5, 9A6and 10A3) are reactive exclusively to the single protein (lane A)corresponding to the purified L-PGDS (lane B), indicating that theseantibodies specifically recognize L-PGDS without reacting to othercontaminants in CSF.

[0142] In addition, the purified L-PGDS was treated with N-glycanase andthen subjected to Western blot analysis. The results are shown in FIG.3. Each lane A shows the sample treated with glycanase, and each lane Bshows the sample not treated with glycanase. As shown in FIG. 3, thereis no significant difference in band density before and after treatmentwith glycanase in the case of 1B7, 6B9, 6F5, 7F5, and 10A3, while in thecase of 9A6, the intensity of the band was significantly increased aftertreatment with glycanase, suggesting that 9A6 recognizes a glycosylationsite or therearound.

[0143] Then, the isotype of each monoclonal antibody was determinedusing a mouse monoclonal antibody typing kit (RPN29, Amersham). Theresults are shown in Table 4. Further, their Kd values toward the E.coli antigen and CSF antigen were determined by the method of Friguet etal. (J. Immunol. Methods, 77, 305-319 (1985)). The results are shown inTable 4. TABLE 4 monoclonal immunoglobulin dissociation constant (nM)antibody subclass E. coli antigen CSF antigen 1B7 IgG₁(λ) 5.4 3.9 6B9IgG₁(κ) >1000 >1000 6F5 IgG₁(λ) 13.2 10.3 7F5 IgG₁(κ) 0.65 4.1 9A6IgG_(2a)(κ) 7.42 >1000 10A3 IgG₁(κ) 0.53 3.9

[0144] 1B7, 6F5, 7F5 and 10A3 showed high affinities for the E. coliantigen and CSF antigen, indicating that these monoclonal antibodiesrecognize the surface of native L-PGDS molecule. 9A6 showed a highaffinity for the E. coli antigen, but it showed least reactivity to theCSF antigen. The results, along with these results in FIG. 3, suggestedthat 9A6 recognizes a glycosylation or therearound. 6B9 showedreactivity in Western blotting as shown in FIG. 2, but showed noreactivity to the E. coli antigen or the CSF antigen, suggesting itsrecognition of the denatured molecule of L-PGDS.

[0145] Then, deletion mutants of L-PGDS were prepared by truncation ofits partial N-terminal amino acid sequence step by step and used asantigens for epitope mapping of their monoclonal antibodies. The PCRprimers used in this experiment are shown below and the results areshown in FIG. 4. The antigens used in this experiment were prepared inessentially the same way as in Example 1(1) except that the L-PGDSproduct was recovered in the form of a fusion protein with GST byboiling in 1% SDS, not by treatment with thrombin.

[0146] forward primer A (amino acids 1 to 7): SEQ ID NO:3.

[0147] forward primer B (amino acids 7 to 12): SEQ ID NO:5.

[0148] forward primer C (amino acids 13 to 18): SEQ ID NO:6.

[0149] forward primer D (amino acids 30 to 35): SEQ ID NO:7.

[0150] forward primer E (amino acids 52 to 57): SEQ ID NO:8.

[0151] forward primer F (amino acids 68 to 73): SEQ ID NO:9.

[0152] forward primer G (amino acids 85 to 90): SEQ ID NO:10.

[0153] forward primer H (amino acids 99 to 105): SEQ ID NO:11.

[0154] forward primer I (amino acids 118 to 123): SEQ ID NO:12.

[0155] forward primer J (amino acids 134 to 139): SEQ ID NO:13.

[0156] forward primer K (amino acids 152 to 158): SEQ ID NO:14.

[0157] reverse primer (amino acids 163 to 168): SEQ ID NO:4.

[0158]FIG. 4 suggested that 7F5 and 10A3 recognize a sequence inAla¹-Val⁶; 9A6, Gln¹³-Asn²⁹; 6F5, Tyr⁸⁵-Val⁹⁸; and 1B7 and 6B9,Gly¹¹⁸-Pro¹³³

[0159] (3) Preparation of Calibration Curves

[0160] The above E. coli antigen was used for preparation of calibrationcurves for L-PGDS. The monoclonal antibodies used were 1B7 and 6F5. A96-well microtiter plate was coated with diluted E. coli antigen andblocked with 0.2% gelatin in PBS. After blocking, the biotinylatedmonoclonal antibody 1B7 or 6F5 was added to each well and incubated.Then, the plate was washed, and a streptoavidin-horseradish peroxidaseconjugate was added to each well and incubated. The plate was washed,and a coloration substrate ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added toeach well. L-PGDS was determined by measuring this coloration by thecalorimetric method. The results are shown in FIGS. 5 and 6 for 1B7 and6F5, respectively.

[0161] Then, the sandwich ELISA method was used for calibration curvesfor L-PGDS. A 96-well microtiter plate was coated with dilutedmonoclonal antibody 1B7 or 6F5 and blocked with 0.2% gelatin in PBS.After blocking, the diluted E. coli antigen was added to each well andincubated. Then, the plate was washed, and the biotinylated polyclonalantibody was added to each well and incubated. Then, the plate waswashed, and a streptoavidin-horseradish peroxidase conjugate was addedto each well and incubated. The plate was washed, and a colorationsubstrate ABTS (2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)was added to each well. L-PGDS was determined by measuring thiscoloration by the calorimetric method. The results are shown in FIGS. 7and 8 for 1B7 and 6F5, respectively. As can be seen from FIGS. 5 to 8,about 10-fold higher sensitivity was attained using the sandwich ELISAmethod.

[0162] In another embodiment of the present invention, the sandwichELISA method and the biotinylated polyclonal or monoclonal antibody wereused for preparation of calibration curves. A 96-well microtiter platewas coated with diluted monoclonal antibody 1B7, 10A3 or 7F5 as primaryantibody and blocked with 0.2% gelatin in PBS. The diluted E. coliantigen was then added to each well and incubated. Then, the plate waswashed, and the biotinylated polyclonal antibody or the biotinylatedmonoclonal antibody 1B7, 7F5 or 10A3 was added as secondary antibody toeach well and incubated. The plate was washed, and astreptoavidin-horseradish peroxidase conjugate was added to each welland incubated. The plate was washed, and a coloration substrate ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added toeach well. L-PGDS was determined by measuring this coloration by thecalorimetric method.

[0163] The results are shown in FIGS. 9 to 11.

[0164]FIG. 9 shows the calibration curves using monoclonal antibody 1B7as primary antibody, where curve A makes use of the biotinylatedpolyclonal antibody as secondary antibody; curve B, biotinylated 7F5 assecondary antibody; and curve C, biotinylated 10A3 as secondaryantibody.

[0165]FIG. 10 shows the resulting calibration curves using monoclonalantibody 10A3 as primary antibody, where curve A makes use of thebiotinylated polyclonal antibody as secondary antibody; and curve B,biotinylated 1B7 as secondary antibody.

[0166]FIG. 11 shows the calibration curves using monoclonal antibody 7F5as primary antibody, where curve A makes use of the biotinylatedpolyclonal antibody as secondary antibody; and curve B, biotinylated 1B7as secondary antibody.

[0167] As can be seen from FIGS. 9 to 11, high sensitivity can beattained using 10A3 or 7F5 as primary antibody and the biotinylatedpolyclonal antibody, or biotinylated monoclonal antibody 1B7, assecondary antibody.

EXAMPLE 3 Detection of L-PGDS Derived from Various Human Tissues

[0168] (1) Detection of L-PGDS in CSF

[0169] A 96-well microtiter plate was coated with diluted monoclonalantibody 1B7 or 6F5 and then blocked with 0.2% gelatin in PBS. Dilutedhuman CSF was then added to each well and incubated. Separately, thepolyclonal antibody prepared using a rabbit as an immunized animal waspurified by affinity column chromatography on Protein A and thenbiotinylated with NHS-LC-Biotinylation Kit (PIERCE).

[0170] Subsequently, the above microtiter plate was washed, and thebiotinylated polyclonal antibody was added to each well and thenincubated. Subsequently, a streptoavidin-horseradish peroxidaseconjugate was added to each well and incubated. The plate was washed,and a coloration substrate ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added toeach well. L-PGDS was determined by measuring this coloration by thecalorimetric method. The results are shown in Table 5. TABLE 5 SampleNo. 1 2 3 4 5 L-PGDS (μg/ml) 23 31 20 10 18

[0171] (2) Detection of L-PGDS in Blood

[0172] L-PGDS in human serum obtained by centrifugation of human bloodwas determined by the same method as in (1) above except that dilutedserum was used. The results are shown in Table 6. TABLE 6 Sample No. 1 23 4 5 L-PGDS (μg/ml) 0.45 0.29 0.21 0.41 0.26

[0173] (3) Detection of L-PGDS in Amniotic Fluid

[0174] L-PGDS in human amniotic fluid was determined by the same methodas (1) above except that diluted amniotic fluid was used. The resultsare shown in Table 7. TABLE 7 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 5.51.2 1.3 2.9 1.8

[0175] (4) Detection of L-PGDS in Semen Supernatant

[0176] L-PGDS in human seminal plasma was determined by the same methodin (1) above except that diluted seminal plasma was used. The resultsare shown in Table 8. TABLE 8 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 4.1 1223 10 8.3

[0177] (5) Detection of L-PGDS in Follicular Fluid

[0178] L-PGDS in human follicular fluid was determined by the samemethod in (1) above except that diluted follicular fluid was used. Theresults are shown in Table 9. TABLE 9 Sample No. 1 2 3 4 5 L-PGDS(μg/ml) 0.21 0.11 0.11 0.15 0.13

[0179] (6) Detection of L-PGDS in Urine

[0180] L-PGDS in human urine was determined by the same method in

[0181] (1) above except that diluted urine was used. The results areshown in Table 10. TABLE 10 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 1.070.65 2.45 1.62 0.32

[0182] (7) Detection of L-PGDS in Various Humors

[0183] The sandwich ELISA system (7F5 monoclonal antibody× biotinylated1B7 monoclonal antibody) established in Example 2(3) was used forquantifying L-PGDS in human CSF, serum and urine. The results (means(±SE)) were compared with those of previous literatures. In this assay,TMBLUE (Intergen-CDP) was used as colouring substrate in place of ABTS.

[0184] The measurement means are shown in Table 11.A, and those of theliteratures are shown in Table 11.B. L-PGDS levels in amniotic fluid andseminal plasma were also determined in the same manner, and the results(means (±SE)) are shown in Table 11.A, as well. TABLE 11.A sample numberof samples means (±SE) (μg/ml) CSF 38 12.07 ± 1.26  serum 12 0.27 ± 0.01urine 10  1.56 ± 0.30* amniotic fluid 52 2.55 ± 0.22 seminal plasma 3213.01 ± 1.72 

[0185] TABLE 11.B number means sample of samples (±SD) (μg/ml)literature CSF 12 40 Pepe,A.J. and Hochwald, G.M. (1967)Proc.Soc.Exp.Biol. 126, 630-633 CSF 59 26(±6) Link,H. and Olsson,J.E.(1972) Acta Neurol.Scadinav. 48, 57-68 CSF 35 27(±1.5) Olsson,J.E.,Link,H., and Müller,R. (1976) J.Neurol. Sci. 27,233-245 CSF 192  33(±11)Felgenhauer,K., Schädlich, H.J., and Nekic,M. (1987) Kiln.Wochenschr.65, 764-768 serum 25 3.9(±0.16) Olsson,J.E., Link,H., and Nosslin,B.(1973) J.Neurochem. 21, 1153-1159 urine 15 3.6˜53.9* Whitsed,H. andPenny,R. (1974) Clin.Chim.Acta 50,119-128

[0186] As shown in the tables, the present means are lower than those ofthe literatures, particularly with respect to serum considered to havecontaminants in abundance. This difference may result from the fact thatL-PGDS might be overestimated in the methods of the literatures becauseof their low specificity, and high specificity in the present assaysystem is clarified.

[0187] A typical calibration curve prepared in these measurements isshown in FIG. 12.

[0188] Curve A is a calibration curve where L-PGDS purified from CSF orthe recombinant L-PGDS prepared in Example 1(1) was used as the standardsubstance. Curves B, C, D, E, and F are calibration curves preparedusing CSF, seminal plasma, urine, amniotic fluid, and serum,respectively. As can be seen from FIG. 12, the sample calibration curvesare nearly parallel with the calibration curve A, so L-PGDS can bespecifically determined in the present assay system without anyinfluence of contaminants in a sample.

[0189] Then, an additive test in the present assay system was carriedout. Human CSF, serum, urine, and seminal plasma were dilutedrespectively to adjust their L-PGDS contents to about 20 ng/ml. PurifiedL-PGDS (CSF antigen) from CSF was added in an amount of 15 ng/ml to eachsample. The results indicated that good recovery (98.5 to 104.6%) wasobtained in every sample as shown in Table 12. TABLE 12 dilution degreeL-PGDS (ng/ml) recovery sample (-fold) initial conc. addition recovery(%) CSF 1 350 20.69 15.00 35.65 99.7 CSF 2 600 21.17 15.00 36.31 100.9serum 1  15 20.71 15.00 36.21 103.3 serum 2  15 13.67 15.00 33.96 101.9urine 1  40 20.86 15.00 36.55 104.6 urine 2  40 18.56 15.00 33.34 98.5seminal 500 16.82 15.00 31.70 99.2 plasma 1 seminal 1000  19.51 15.0034.93 102.8 plasma 2

EXAMPLE 4

[0190] L-PGDS in 5 samples (seminal plasma) was detected by Westernblotting using the monoclonal antibody 1B7. The results are shown inFIG. 13. As shown in FIG. 13, L-PGDS was detected specifically,indicating that this monoclonal antibody is reactive exclusively toL-PGDS in the presence of other contaminants.

[0191] Then, the sandwich ELISA system (monoclonal antibody 1B7×biotinated polyclonal antibody) established in Example 2 was used toquantify L-PGDS in 1 ml seminal plasma from each of healthy normalpersons (40 cases) and patients with oligospermia (10 cases). The numberof spermatozoa in 1 ml semen was determined with a Makler countingchamber. The results are shown in Table 13. number of spermatozoaL-PGDS(μg/ml) sample no. (×10⁴ spermatozoa/ml) subject by ELISA 1   0oligospermia patient 2.6 2  100 oligospermia patient 0.8 3  100oligospermia patient 1.0 4  800 oligospermia patient 3.8 5  800oligospermia patient 2.2 6 1000 oligospermia patient 0.9 7 1800oligospermia patient 1.0 8 2000 oligospermia patient 5.0 9 2000oligospermia patient 4.8 10 2000 oligospermia patient 2.6 11 2500healthy person 4.3 12 3000 healthy person 7.5 13 3000 healthy person23.6 14 3000 healthy person 30.0 15 3000 healthy person 9.5 16 3000healthy person 2.5 17 3000 healthy person 4.4 18 4000 healthy person 6.019 4000 healthy person 30.0 20 4200 healthy person 8.0 21 5000 healthyperson 1.9 22 6000 healthy person 42.0 23 6000 healthy person 1.5 246000 healthy person 3.8 25 6500 healthy person 2.0 26 7000 healthyperson 3.2 27 7000 healthy person 8.2 28 8000 healthy person 9.6 29 8000healthy person 14.0 30 8500 healthy person 6.0 31 8800 healthy person3.0 32 9000 healthy person 8.0 33 9000 healthy person 7.1 34 10000 healthy person 10.1 35 10000  healthy person 3.0 36 10000  healthyperson 7.5 37 10000  healthy person 6.0 38 11000  healthy person 0.3 3912000  healthy person 17.0 40 12000  healthy person 2.6 41 12000 healthy person 7.0 42 12000  healthy person 1.1 43 13000  healthy person10.5 44 13900  healthy person 30.0 45 15000  healthy person 16.0 4615000  healthy person 5.1 47 15000  healthy person 3.5 48 16000  healthyperson 13.0 49 17400  healthy person 15.0 50 18000  healthy person 6.2

[0192] The results indicated that L-PGDS levels are 9.75±1.486 μg/ml(healthy persons) and 2.470±0.509 μg/ml (oligospermia patients), and thedifference therebetween is statistically significant at P≦0.0008 level(Mann-Whitney method) or P<0.0192 level (Fisher method), in FIG. 14.Because many samples can be dealt with in a short time using the kit ofthe present invention, it is useful for diagnosis of oligospermia.

Example 5

[0193] One-step purification of L-PGDS from CSF was attempted bycoupling each of monoclonal antibodies 1B7, 6F5, 9A6, 7F5 and 10A3 tocarriers by use of an Affi-Gel Hz immunoaffinity kit (Bio-Rad).

[0194] First, 10 ml gel with each monoclonal antibody coupled theretowas equilibrated with PBS. A CSF solution, prepared by diluting 10 ml ofCSF at least 3-fold with PBS, was applied to the gel. The gel was washedwith 20 ml PBS containing 2 M NaCl, then 30 ml PBS containing 0.1%Triton X-100, and finally with 50 ml PBS, and the protein was elutedwith 0.1 M sodium citrate (pH 3.0). Using any kind of antibody, L-PGDScan be efficiently adsorbed onto the gel and eluted with 0.1 M sodiumcitrate (pH 3.0), and the resulting preparation having enzymaticactivity was almost homologous in SDS-PAGE. The yield was about 80% andthe product was purified as high as 37-fold relative to the originalCSF.

[0195] One Example of such purification is shown in FIG. 15.

[0196] Industrial Applicability

[0197] According to the present invention, there is provided themonoclonal antibody specific to L-PGDS.

[0198] The analysis of the distribution of L-PGDS in the central nervoussystem is useful for detection of diseases in the central nervoussystem, and it is expected that L-PGDS levels in CSF or humor can alsobe used as an indicator in early diagnosis and prognostic observationsfor other diseases caused by abnormalities in the central nervoussystem. It is further expected that L-PGDS (βtrace) can be used forexamination of a reproduction ability, diagnosis of fetal growth, etc.because this enzyme is distributed in such humors derived from genitalorgans, as semen, oviduct fluid and amniotic fluid, as well. Further, itwas recently revealed in our study that the gene for L-PGDS is expressedin the heart too and therefore the distribution and levels of L-PGDS inblood and other humors can also be used for diagnosis of diseases in thecirculatory organs.

[0199] Accordingly, the monoclonal antibody provided according to thepresent invention is useful as a reagent in studying the expression,tissue distribution, physiological action etc. of L-PGDS, and as areagent for pathological diagnosis of various diseases in the centralnervous system as well as in the genital and circulatory organs.

1 15 1 22 DNA Artificial Sequence primer 1 gggaattcat gcacccgagg cc 22 220 DNA Artificial Sequence primer 2 gaggtcaggg cgaagccacc 20 3 29 DNAArtificial Sequence primer 3 ccggatccgc acccgaggcc caggtctcc 29 4 30 DNAArtificial Sequence primer 4 atgaattcac tattgttccg tcatgcactt 30 5 26DNA Artificial Sequence primer 5 ccggatcctc cgtgcagccc aacttc 26 6 26DNA Artificial Sequence primer 6 ccggatccca gccggacaag ttcctg 26 7 26DNA Artificial Sequence primer 7 ccggatcctc gagctggctc caggag 26 8 26DNA Artificial Sequence primer 8 ccggatccga tggtggcttc aacctg 26 9 26DNA Artificial Sequence primer 9 ccggatccga gacccgaacc atgctg 26 10 25DNA Artificial Sequence primer 10 ccggatccta ccggagtccc cactg 25 11 27DNA Artificial Sequence primer 11 ccggatccgt ggagactgac tacgacc 27 12 26DNA Artificial Sequence primer 12 ccggatccgg cgaggacttc cgcatg 26 13 26DNA Artificial Sequence primer 13 ccggatccag ggctgagtta aaggag 26 14 29DNA Artificial Sequence primer 14 ccggatccga ggattccatt gtcttcctg 29 15168 PRT Homo sapiens 15 Ala Pro Glu Ala Gln Val Ser Val Gln Pro Asn PheGln Pro Asp Lys 1 5 10 15 Phe Leu Gly Arg Trp Phe Ser Ala Gly Leu AlaSer Asn Ser Ser Trp 20 25 30 Leu Gln Glu Lys Lys Ala Ala Leu Ser Met CysLys Ser Val Val Ala 35 40 45 Pro Ala Ala Asp Gly Gly Phe Asn Leu Thr SerThr Phe Leu Arg Lys 50 55 60 Asn Gln Cys Glu Thr Arg Thr Met Leu Leu GlnPro Gly Asp Ser Leu 65 70 75 80 Gly Ser Tyr Ser Tyr Arg Ser Pro His TrpGly Ser Thr Tyr Ser Val 85 90 95 Ser Val Val Glu Thr Asp Tyr Asp His TyrAla Leu Leu Tyr Ser Gln 100 105 110 Gly Ser Lys Gly Pro Gly Glu Asp PheArg Met Ala Thr Leu Tyr Ser 115 120 125 Arg Thr Gln Thr Pro Arg Ala GluLeu Lys Glu Lys Phe Thr Ala Phe 130 135 140 Cys Lys Ala Gln Gly Phe ThrGlu Asp Ser Ile Val Phe Leu Pro Gln 145 150 155 160 Thr Asp Lys Cys MetThr Glu Gln 165

1. A monoclonal antibody specifically recognizing lipocalin-typeprostaglandin D synthase (L-PGDS).
 2. The monoclonal antibody accordingto claim 1, wherein the subclass of said monoclonal antibody isimmunoglobulin G1 or G2.
 3. A hybridoma obtained by cell fusion betweenantibody-producing cells from an animal immunized with L-PGDS andmyeloma cells and producing a monoclonal antibody of claim 1 or
 2. 4. Amethod for detection of L-PGDS, wherein L-PGDS is detected by amonoclonal antibody of claim 1 or
 2. 5. A method for detection of adisease, wherein L-PGDS is detected by a monoclonal antibody of claim 1or
 2. 6. The method according to claim 5, wherein the disease isoligospermia.
 7. A kit for detection of L-PGDS, which is selected fromthe group consisting of: a reagent containing the monoclonal antibody ofclaim 1 or 2 labeled with an enzyme, and a substrate solution; a reagentcontaining the monoclonal antibody of claim 1 or 2, said monoclonalantibody being labeled with an enzyme, or an enzyme-labeled polyclonalantibody recognizing human L-PGDS, and a substrate solution; a reagentcontaining the monoclonal antibody of claim 1 or 2 which isbiotinylated, enzyme-labeled avidin or streptoavidin, and a substratesolution; and a reagent containing the monoclonal antibody of claim 1 or2, said monoclonal antibody being biotinylated, or a biotinylatedpolyclonal antibody recognizing human L-PGDS, enzyme-labeled avidin orstreptoavidin, and a substrate solution.
 8. A method for detection of adisease, wherein the disease is detected using the kit for detection ofL-PGDS described in claim
 7. 9. The method according to claim 8, whereinthe disease is oligospermia.